Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance in a fluid. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer or detection reagent. Immunoassay procedures may be carried out in solution or on a solid support and may be either heterogeneous, requiring a separation of bound tracer from free (unbound) tracer or homogeneous in which a separation step is not required.
Enzymes have often been used as labels in immunoassays. In a conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. The signal may be a color change, detected with the naked eye or by a spectrophotometric technique, or may be conversion of the substrate to a product detected by fluorescence.
The enzymes that are used as labels in an immunoassay must be stable, highly active, available in a highly purified form, yield stable conjugates and be inexpensive, safe and convenient to use. An enzyme which meets these criteria and is extensively used in immunoassays is alkaline phosphatase (hereinafter referred to as "AP"). AP catalyzes the cleavage of phosphate groups from generally colorless phosphorylated substrates to give colored products.
A typical assay is a sandwich immunoassay for detecting an antigen. In this assay, a capture antibody is affixed to a solid support such as a dipstick, membrane, microparticles, microtiter plate well or the inside wall of a tube. The antibody-coated solid support is further coated with an inert protein, such as casein or albumin to block substantially all remaining binding sites on the support and thereby suppress nonspecific binding of tracer directly to the support. Blocking with an inert protein is conventional in the immunoassay art.
A sample solution suspected of containing an antigen is added to the antibody coated and blocked support and conditions conducive to binding the antigen to the antibody are provided. A tracer including a second antibody labeled by covalent conjugation to AP is added. After binding of the second antibody to the antigen, the solid support having affixed thereto an antibody-antigen-labeled antibody bound fraction is contacted with a substrate of AP. The AP substrate is dephosphorylated by the AP component of the bound tracer on the solid support to form a color. The color is indicative of the presence of the antigen and the intensity of the color is directly proportional to the concentration of the antigen in the liquid.
In a typical competitive assay, a limited quantity of the antibody on the solid support may be contacted with the sample and a tracer which includes a known quantity of the antigen having AP conjugated thereto. The antigen and AP-labeled antigen bind to the antibody on the support in direct proportion to their concentrations in the solution. Thus, after binding, the support contains an antibody-antigen bound fraction and an antibody-AP-labeled antigen bound fraction. After separation of the support from the assay solution, the bound fractions on the support may be contacted with the AP substrate to cause formation of a color. However, in the competitive assay of the invention, the color formed is inversely proportional to the concentration of antigen in the liquid.
Endogenous AP is also found in clinical samples and it may interfere with immunoassays using exogenous AP as labels. Typically, in mammals, AP exists in different forms as different isoenzymes. On the basis of tissue specificity, human AP isoenzymes are classified into three types: tissue-nonspecific (found in liver, kidney, bone, spleen, etc.), intestinal, and placental types. These isoenzymes have been well characterized by enzymological and immunochemical approaches {Fishman, W. H. (1974) Am. J. Med. 56, 617-650}. Recently, cDNAs for tissue-nonspecific {Weiss, M. J., et al, (1986) Proc. Natl. Acad. SicU.S.A. 83, 7182-7186}, intestinal {Berger, J. et al., (1987) Proc. Natl. Acad. SicU.S.A. 84, 695-698}, and placental {Kam, W., et al., (1985) Proc. Natl. Acad. SicU.S.A. 82, 8715-8719; Millan, J. L. (1986) J. Bio. Chem. 261, 3112-3115; Henthorn, P. S., et al., (1986) Proc. Natl. Acad. SicU.S.A. 83, 5597-5601} isoenzymes have been isolated and these have helped in the understanding of the difference and homology in the primary structures of the three isoenzymes.
Phenylalanine and levamisole, (L-)2,3,5,6-tetrahydro-6-phenylimidazo[1,2-b]thiazole, are two well-known inhibitors of AP. The tissue-nonspecific AP are not sensitive to L-phenylalanine, but are strongly inhibited by levamisole; whereas the placental and intestinal isoenzymes are inhibited by both L-phenylalanine and levamisole but only at much higher concentrations.
Advantage has been taken of the above selectivity of the inhibitors in immunoassays (see, e.g., U.S. Pat. No. 5,093,231). When used in an assay, levamisole does not interfere with the specific immuno-signal generated by calf intestinal AP but does reduce the nonspecific signal which arises from any non-intestinal AP which may be present in a clinical sample. Morris et al., in the Journal of Immunological Methods 68, 11 (1984) disclose detection of the binding of monoclonal antibodies to antigens on the surface of whole cells with a conjugate of calf intestinal AP and goat anti-mouse antibodies in the presence of a substrate and levamisole added to inhibit AP of non-intestinal origin.
Ponder et al., (1981) Journal of Histochemistry and Cytochemistry 29, 981, disclose detection of mouse H2 antigen in tissue slices by incubating the tissue slices with anti mouse H2 antibody and treating with a calf intestinal AP labeled conjugate followed by a substrate for the enzyme and levamisole to inhibit non intestinal AP. In the Ponder et al. method, levamisole at a concentration of 1 mM is added to a filtered AP substrate solution prior to combining the substrate with the tissue slices.
Levamisole is also used as an antihelminthic drug (U.S. Pat. No. 4,137,321 to Leeming, et al.; U.S. Pat. No. 4,143,147, to Leeming, et al.; U.S. Pat. No. 4,389,406 to Dorgan et al.; U.S. Pat. No. 4,370,482 to Raghu, et al.; U.S. Pat. No. 4,310,672 to Raghu, et al.; U.S. Pat. No. 4,139,707 to Raghu, et al.; and U.S. Pat. No. 4,090,025 to Raghu et al.).
Levamisole has also been used as a nonspecific immunomodulator in the adjuvant treatment of various malignancies (U.S. Pat. No. to 4,584,305 to Brugmans et al.). Levamisole was approved in June 1990 by the United States Drug and Food Administration under the name ERGAMISOL, and is sold by Janssen Pharmaceutical for use in combination with fluorouracil, an already approved drug, for adjuvant treatment of stage C colon cancer after surgical resection.
Levamisole combined with fluorouracil has been associated with one-third reduction in recurrence and risk of death in patients with surgically resected stage C colon cancer as described in C. G. Moertel et al., New Eng. J. Med. 322, 352-358 (1990).
Levamisole has also been described as an antidepressive agent (U.S. Pat. No. 3,852,458 to Janssen) and as an antianergic agent (DE 2340633 assigned to Janssen Pharmaceutical). It is also described as possessing psychoenergising and anti-anorexigenic activities, U.S. Pat. No. 4,005,212 to Debarre et al.